MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
Biosens Bioelectron. 2012 Jan 15;31(1):463-8. doi: 10.1016/j.bios.2011.11.016. Epub 2011 Nov 17.
Here we have developed a novel nano-magnetic primer based electrochemiluminescence-polymerase chain reaction (NMPE-PCR) strategy for detection of genome. The key idea of this method is integrating the two in situ processes: PCR on the surface of magnetic nanoparticles (MNPs) and magnetic beads based ECL readout platform, to avoid some laborious manual operations and achieve rapid yet sensitive detection. At first, the approach employs a pair of functional primers for amplification: one is tris-(2,2'-bipyridyl) ruthenium (TBR) labeled primer; the other one is nano-magnetic primer which is prepared by attaching the primer to the surfaces of MNPs. With the presence of DNA analyte and PCR mixture, the TBR labeled products are directly loaded and enriched on the surface of MNPs during PCR cycling. Then the MNPs-TBR complexes can be analyzed by a magnetic ECL platform without any post-modification or post-incubation. Finally, we used Listeria monocytogenes as the target to examine these desirable properties of this assay, reaching a detection limit of 500 fg/μL for genome in 1 h. The proposed study has provided the evidence as a proof-of-concept, thus having potential for development of automatic mode for detection of specific gene.
在这里，我们开发了一种基于新型纳米磁引物的电化学发光-聚合酶链反应（NMPE-PCR）策略来检测基因组。该方法的关键思想是整合两个原位过程：在磁性纳米颗粒（MNPs）表面上的 PCR 和基于磁珠的 ECL 读取平台，以避免一些繁琐的手动操作并实现快速而灵敏的检测。首先，该方法采用一对功能引物进行扩增：一个是三（2,2'-联吡啶）钌（TBR）标记的引物；另一个是纳米磁引物，它是通过将引物附着在 MNPs 表面上制备的。在存在 DNA 分析物和 PCR 混合物的情况下，TBR 标记的产物在 PCR 循环过程中直接加载并富集在 MNPs 表面上。然后，无需任何后修饰或后孵育，即可通过磁 ECL 平台分析 MNPs-TBR 复合物。最后，我们使用单核细胞增生李斯特菌作为靶标来检验该测定法的这些理想特性，在 1 小时内达到 500 fg/μL 基因组的检测限。该研究提供了概念验证的证据，因此具有开发用于特定基因检测的自动模式的潜力。
